Abstract The bacterial cell envelope is a remarkable and complex structure that guards bacteria from their surrounding environment. A defining feature of gram-negative bacteria is the presence of an outer membrane (OM) that encapsulates the peptidoglycan layer of these organisms. While the inner membrane (IM) is composed of glycerophospholipids (GPLs), the OM is a bilayer with extreme lipid asymmetry with GPL confined to the inner leaflet and lipopolysaccharide (LPS) localized to the outer leaflet. This unique membrane organization affords gram-negative bacteria protection from large polar molecules, as well as lipophilic compounds, serving as an essential innate barrier to a variety of antibiotics. However, once assembled, environmental factors can disrupt the LPS monolayer resulting in shedding of LPS, and as a consequence, migration of GPLs from the inner leaflet to the outer leaflet of the OM. Extensive formation of GPL rafts at the bacterial surface results in the loss of barrier function which leads to cell death. To prevent this, the cell must maintain the OM asymmetry even under extreme environmental stress. The overall objective of this application is to investigate the molecular mechanisms required for maintenance of OM asymmetry, including the role of the recently identified Mla retrograde GPL transport system. We will also investigate two additional systems, Pqi and Yeb systems, that may also serve in GPL transport. All three systems (Mla, Pqi, and Yeb) are highly conserved across Gram-negative bacteria and disruption of OM maintenance machinery has been shown to result in decreased virulence for many pathogens. The Specific Aims of the current proposal are: (1) structure and functional analysis of Mla lipid binding proteins; (2) overall architecture and protein-protein interactions of Mla components; and (3) investigation of lipid binding by GPL transport systems in whole bacteria. Completion of the Aims will fill major gaps towards understanding maintenance of OM asymmetry and provide new avenues for the generation of novel antimicrobials.